Characterization of a T-Lymphocyte Epstein-Barr Virus/C3dReceptor (CD21)
JOYCE D. FINGEROTH,1* MARTHA L. CLABBY,2 AND JACK D. STROMINGER2
Divisions ofInfectious Diseases' and Tumor Virology,2 Dana-Farber Cancer Institute, Boston, Massachusetts 02115
Received 24 September 1987/Accepted 10 December 1987
The Epstein-Barr virus/C3d receptor (EBVR-CR2) was detected on three T-lymphoblastoid cell lines. Theapparent Mrs of purified EBVR-CR2 of T-cell and B-cell origin were identical. The N-terminal amino acidsequence from the T-cell EBVR-CR2 confirmed the placement of this receptor in a multigene family ofcomplement regulatory proteins. All EBVR-CR2-positive T-cell lines were T6 and T4-T8 antigen positive.
Epstein-Barr virus (EBV), a human herpesvirus, causesinfectious mononucleosis and polyclonal B-cell lymphoma(7). EBV is associated with African Burkitt's lymphoma andnasopharyngeal carcinoma (7). The human B-lymphocytereceptor for EBV is a 145,000-Mr glycoprotein (8, 9, 37) thatis also the receptor for the d region of C3 (CR2) (17, 55). Arole for this receptor in normal B-lymphocyte activation hasbeen postulated (10, 15, 29, 36, 49, 57). Internal amino acidsequences derived from tryptic peptides and a partial cDNAclone demonstrates that EBV CR2 (EBVR-CR2) is homolo-gous to complement receptor type 1 (CR1) (19, 54), whichplaces it in a multigene family (38) that includes the comple-ment regulatory proteins C4-binding protein (5), factor H(21, 41), and decay-accelerating factor (4, 28), all five ofwhich are linked on chromosome 1 (27, 42, 53). Additionalcomplement proteins which share a characteristic 60-amino-acid consensus repeat and concomitant serine proteaseactivity include factor B (11, 32), C2 (11), and Clr-Cls (24,45). The noncomplement proteins beta-2 glycoprotein 1 (26),the beta subunit of factor XIII (16), the alpha' chain ofhaptoglobin (22), and the interleukin-2 receptor (23) are alsomembers of this extended multigene family (38).EBVR-CR2 was originally found on B lymphocytes with
the exception of the T-lymphoblastoid cell line Molt 4 (8, 30).Immunohistochemical staining procedures suggest that thereceptor may also be expressed on follicular dendritic cells(40), histiocytosis X cells (1), and a subset of oropharyngealepithelial cells (58). Identity of EBVR-CR2 with cross-reactive antigens on these distinct cell types has not beenestablished by biochemical means or functional studies.EBVR-CR2 (CD21) was purified from (13, 18) a B-lym-
phoblastoid cell line (JY) and from a T-lymphoblastoid cellline (HPB-ALL) by lectin (18) and immunoaffinity chroma-tography (44). To purify the receptor, membrane lysates (36,37) in detergent were passed over three serial lectin columns(ricin, lentil, and wheat germ) which were eluted withappropriate sugars (37); the lysates were then pooled in thepresence of sodium deoxycholate (final concentration,0.5%), and this lysate pool was applied to an immunoaffinitycolumn of anti-HB-5 MAb (40), which was washed aspreviously described (37). The column was preeluted with 1column volume of 50 mM diethylamine (pH 11.5) (FisherScientific Co.)-0.1% sodium deoxycholate (which removedlittle of the EBVR-CR2 [<2%] but rapidly cleared thecolumn of minor contaminants), washed with 2 columnvolumes of 10 mM Tris (pH 7.5, 0.1%), and finally eluted in
* Corresponding author.
2 to 3 column volumes of 3 M KSCN-10 mM Tris (pH7.5)-0.1% sodium deoxycholate. Separate columns wereused for the respective cell lines throughout the purification.The eluted protein was dialyzed against 10 mM Tris-0.04 MNaCl-0.1% sodium deoxycholate, precipitated with 5 vol-umes of cold acetone, suspended in 500 ,ul of water, andanalyzed for yield and purity. The molecular weights ofhighly purified proteins from JY and HPB-ALL appeared tobe identical when assessed by silver stain (33) of the purifiedreceptor run on sodium dodecyl sulfate-polyacrylamide gelsunder reducing (Fig. 1) and nonreducing conditions. Asimple and rapid purification scheme yielded an average of0.5 to 3 ,ug of pure protein per g of cells from 50- to 100-gbatches.
Purified EBVR-CR2 was precipitated in ethanol, dried,suspended in 100 ,ul of water-0.01% recrystallized sodiumdodecyl sulfate, and sequenced on a gas-phase sequenator(18). The N-terminal sequence from 1 to 2 nmol of purifiedHPB-ALL EBVR-CR2 was obtained on two occasions(Table 1). No N-terminal amino acid sequence was obtainedfrom 1 nmol of reduced and alkylated JY receptor, suggest-ing qualitative or quantitative alterations in the proteinpreparation or amino acid substitution in the B-cell proteinresulting in N-terminal cyclization or other biochemicalmodification that inhibited the sequencing reaction. TheN-terminal amino acids of HPB-ALL EBVR-CR2 werehomologous to internal peptides from the B-cell protein(Table 1). Identification of the consensus repeat at theextreme N terminus was consistent with the structure ofseveral members of the 60-amino-acid-repeat family, includ-ing C4-binding protein, factor H, decay-accelerating factor,beta-2 glycoprotein I, the b subunit of factor XIII, andprobably CR1 (19), whose members share a similar organi-zation, with tandem repeats proceeding from or near theamino end of the molecule and terminating in a shortC-terminal domain (38) (Table 1). The N-terminal portions ofselected consensus sequences from more structurally di-verse family members (Table 1), including factor B, C2,Clr-Cls, factor I, the alpha' chain of haptoglobin, and theinterleukin-2 receptor, also demonstrate significant similar-ity at positions 8, 10, 12, and 13 beyond the absoluteconservation at positions 4 and 7. Secondary structureanalysis of the EBVR-CR2 N terminus by a modified Chou-Fasman pseudoprobabilities code (37a) reveals a beta turnoriginating close to or at the N terminus, followed by a smallbeta pleated sheet. Because the C3b-C4b binding domains ofC4-binding protein, factor H, the C2b fragment of C2, andthe Ba fragment of factor B have been localized to their
1442
Vol. 62, No. 4
NOTES 1443
1 2 3 4 5 6
200-
145-
116-
97-
FIG. 1. Silver-stained 7.5% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Lanes 1 to 3 contain serial dilutions ofpurified EBVR-CR2 from the T-lymphoblastoid cell line HPB-ALL.Lanes 4 to 6 contain serial dilutions of purified receptor protein fromthe JY cell line. Numbers on the left indicate molecular weights ofstandard proteins (the purified protein is at 145).
respective N termini (38), it will be interesting to determinewhether EBVR-CR2, which is distinguished by binding C3dand EBV, also contains N-terminal sequences important forligand attachment.The observation that EBVR-CR2 was expressed on the
T-cell line HPB-ALL as well as on Molt 4 (8, 30) (Fig. 2)prompted us to examine several additional T lymphocytes.
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Two monoclonal antibodies (MAbs), HB-5 (50) and anti-B2(34), with specificity for distinct epitopes on EBVR-CR2were employed. HB-5 was prepared by 45% ammoniumsulfate precipitation of murine ascites obtained followingintraperitoneal injection of 107 HB-135 hybridoma cells(American Type Culture Collection). Anti-B2 was purchasedfrom Coulter Immunology, as was the control MAb anti-Bl(34). The MAb EBVCS (48) was a gift of Bill Sugden. The7F.10 MAb directed to T6 (CD1) was provided by NancyJones. The anti-CR1 MAb anti-C3bR was purchased fromDako, and the irrelevant MAb P3 was provided by MartinHemler. Fluorescein isothiocyanate (FITC)-coupled goatF(ab')2 anti-mouse immunoglobulins G, A, and M were
purchased from Organon Teknika. EBV was purified fromthe B-958 cell line and fluoresceinated as described previ-ously (8). Established cell lines were maintained as de-scribed previously (8). For staining, samples containing 5 x105 cells each were washed twice with RPMI-5% heat-inactivated fetal calf serum, incubated with antibody 1 for 30min on ice, washed twice, reincubated with FITC-labeledantibody 2 for 30 min on ice, washed twice more, and eitherfixed with 1% paraformaldehyde in phosphate-buffered sa-
line or analyzed immediately following the addition of 10 plof propidium iodide at 0.5 mg/ml. Duplicate samples werestained with FITC-EBV as described elsewhere (8). Flowcytometry was performed on an EPICS V (Coulter) duallaser cell sorter with an MDADS data acquisition package.
Analysis of cells stained with HB-5, anti-B2, and FITC-EBV indicated that the T-cell line Jurkat also expressedEBVR-CR2, while the T-cell line T-ALL-1 did not (Fig. 2).Incubation with HB-5 and anti-mouse immunoglobulinblocked binding of FITC-EBV on all receptor-bearing lines
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JY 17 (not shown) and for the T-cell lines HPB-MLT 26, Jurkat 5 (not shown), Molt 3 5, T-ALL-1 0, and HSB-2 0 (not shown). To obtainthese values, the mean channel fluorescence in arbitrary logarithmic units was calculated from data obtained with MAb HB-5 andFITC-labeled goat F(ab')2 anti-mouse immunoglobulins G, A, and M (Organon Teknika) and converted to a linear scale, and the value of thecontrol antibody, P3, was subtracted. Fluorescence was achieved with 500 mW of 488-nm light from an argon ion laser. A 525-nm bandpass-560-nm short-pass filter combination was used for detection.
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examined, as previously demonstrated (8). The rank order ofmean channel fluorescence for these T-lymphoblastoid celllines was HPB-ALL (also called HPB-MLT) > Jurkat (alsocalled JM) > Molt 3 (from the same patient as Molt 4) (seethe legend to Fig. 2). The mean fluorescence intensity of theHPB-ALL cell line was comparable to that of the B-cell lineJY but was less intense than that of Raji (see the legend toFig. 2), suggesting approximately equivalent receptor densi-ties among receptor-positive T cells compared with B-cells,i.e. 8,000 to<70,000 receptors per cell (8). EBVR-CR2-positive T-cell lines HPB-ALL and Jurkat also expressedCR1, while the cell line Molt 3, which expressed low levelsof EBVR-CR2, did not have CR1 detectable by flow cytom-etry. The B-cell-specific antigens Bi and EBVCS (CD23)(Blast-2) (Fig. 2), the latter an antigen present on manyB-lymphoblastoid cell lines and associated with EBV trans-formation (48, 51, 52), were absent on all T-lymphoblastoidcell lines. Phenotypic analysis of three EBVR-CR2-bearingT-lymphoblastoid cell lines revealed that they all expressedT6 (CD1) (3) (Fig. 2) as well as both T4 and T8 cell surfaceantigens and were HLA-DR negative (31). No EBVR-CR2was detected on cloned T4- and T8-positive cells fromperipheral blood. Southern blot analysis of EBVR-CR2-positive T-cell DNA, with the EBV BamHI W genomicfragment used as a probe, indicated that the cells did notharbor endogenous virus.
Previous identification at low density of an EBVR-CR2 (8,30) ostensibly incapable of internalizing virus (30) on theT-cell line Molt 4 was regarded as an unusual event related tothe transformed state of this tumor. Several T-cell tumorlines previously examined, including HSB-2, Hut-78, CEM,and T-ALL-1, were EBVR-CR2 negative. However, theT-cell line HPB-ALL expressed receptors at a much higherdensity than that observed on several B-lymphoblastoidcells, while the T-cell line Jurkat expressed low levels ofEBVR-CR2. No other B-cell-specific markers were found onthese T cells. However, all the cell lines identified were T6and also T4-T8 antigen positive. A fresh T-lymphoid neo-plasm of thymic phenotype stained with the MAb OKB7 alsodirected to EBVR-CR2 was previously described (20). Al-though it was reported that T6-positive cells from normalthymus are EBVR-CR2 negative (50), the recently definedcomplexity of the CD1 antigen family of proteins includingT6 (3) suggests that reexamination of receptor expressionduring thymic ontogeny could prove interesting. T6-positiveT lymphocytes have been reported to circulate transiently inthe peripheral blood of burn patients (J. Wood, J. B.O'Mahoney, S. B. Palder, M. L. Rodrick, P. O'Eon, andJ. D. Mannick, Letter, J. Invest. Dermatol. 82:387-388,1984) and patients treated for Wiskott-Aldrich syndrome(39). Very rare T6-positive lymphocytes have also beenidentified in normal cord blood and peripheral blood (6, 14).Of interest in this regard is a recent report of an EBV-genome-positive thymoepithelioma (25) and also of an EBV-transformed T-cell line from cord blood (47). These reportssupport the notion that rare T lymphocytes expressingEBVR-CR2 may exist in vivo and may be susceptible totransformation by EBV. Increased rates of spontaneousrecombination (2) and trisomy associated with myeloid ma-lignancy (43) have been ascribed to the complement regula-tory protein locus, raising the possibility that a direct geneticalteration accounts for EBVR-CR2 expression in the T-cellneoplasms identified. Interestingly, the CD1 multigene fam-ily also maps to chromosome 1 (3); however, its location inrelation to the complement regulatory protein locus is un-known. The significance of concurrent expression of T6 and
T4-T8 antigens on EBVR-CR2-positive T-cell neoplasmsremains to be determined.B cells which harbor virus but continue to express recep-
tors can bind exogenous EBV, and genome-negative recep-tor-bearing B-lymphoblastoid lines can be converted tolatent genome-positive cell lines by superinfection withactive virus (46). The tropism of EBV for the human Blymphocyte has been explained on the basis of receptorspecificity. However, the process of virus internalizationand establishment of latency is more complex. InfectedB-lymphoblastoid cell lines do not internalize virus by thesame pathway as virgin B lymphocytes from peripheralblood (12, 35, 49). Possible explanations for this observationinclude interferon effects on the cell membrane such as thosedescribed for other viruses (56), specific EBV-mediated cellmembrane alterations secondary to latent viral proteins or toactivated cellular proteins, or, in the case of FBV-negativetumor lines, membrane alterations otherwise associated withthe transformed state. Identification of the virus receptor onseveral EBV-negative T-lymphoblastoid cell lines allows oneto approach the question of whether a functional receptor isboth necessary and sufficient for the establishment of'latentEBV infection or whether the B-cell milieu itself is requiredfor internalization-fusion or the establishment of latent ver-sus lytic infection. Comparison of different pathways ofinfection should prove informative.
We thank Nancy Jones for helpful discussions and David An-drews and William Lane for N-terminal amino acid sequencing.
This work was supported by Public Health Service grantsK08CA01085 and 5P01CA21082 from the National Institutes ofHealth.
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